Mobilization of the iron centre in IscA for the iron–sulphur cluster assembly in IscU

Abstract

The biogenesis of iron-sulphur clusters requires the co-ordinated delivery of both iron and sulphur. It is now clear that sulphur in iron-sulphur clusters is derived from L-cysteine by cysteine desulphurases. However, the iron donor for the iron-sulphur cluster assembly still remains elusive. Our previous studies indicated that Escherichia coli IscA, a member of the iron-sulphur cluster assembly machinery, is an iron-binding protein that can provide iron for the iron-sulphur cluster assembly in a proposed scaffold IscU. To determine how the iron centre in IscA is transferred for the iron-sulphur cluster assembly in IscU, we explore the mobility of the iron centre in IscA. The UV-visible and EPR measurements show that L-cysteine, but not IscU, is able to mobilize the iron centre in IscA and make the iron available for the iron-sulphur cluster assembly in IscU. Other related biological thiols such as N-acetyl-L-cysteine or reduced glutathione have no effect on the iron centre of IscA, suggesting that L-cysteine is unique in mobilizing the iron centre of IscA. Nevertheless, L-cysteine alone is not sufficient to transfer the iron from IscA to IscU. Both L-cysteine and cysteine desulphurase (IscS) are required for the IscA-mediated assembly of iron-sulphur clusters in IscU. The results suggest that L-cysteine may have two distinct functions in the biogenesis of iron-sulphur clusters: to mobilize the iron centre in IscA and to provide sulphur via cysteine desulphurase (IscS) for the iron-sulphur cluster assembly in IscU.

Controlled Expression and Functional Analysis of Iron-Sulfur Cluster Biosynthetic Components within Azotobacter vinelandii

“…The function of IscA has not been firmly established. However, on the basis of biochemical studies, it has been proposed to serve as either a scaffold for [Fe-S] cluster formation or an agent of Fe delivery to the IscU scaffold (10,24,29,43,64). In the present work, a clear null growth phenotype was manifested in cells depleted of IscA only when challenged by elevated levels of oxygen.…”

ErpA, an iron–sulfur (Fe–S) protein of the A-type essential for respiratory metabolism in Escherichia coli

“…Hence, we might expect a great diversity in the way bacteria make use of otherwise conserved components to carry out Fe-S biogenesis. Another challenge to the view of A-type proteins as scaffolds comes from in vitro investigations, which concluded that A-type proteins could be simple iron donors (30)(31)(32)(33)(34). Interestingly, the erpA gene is part of a chromosomal region that is particularly rich in iron uptake-related genes.…”

“…Three conserved Cys residues could act as ligands for these clusters, as indicated by mutagenesis studies and by the recently obtained 3D structure of IscA (26)(27)(28)(29). However, the proposal that IscA/SufA acts as a scaffold was challenged by in vitro studies, which suggest that A-type proteins act as iron chaperones, and by the fact that mutations in iscA or sufA did not yield major physiological defects (30)(31)(32)(33)(34)(35)(36). Noticeably, however, in A. vinelandii, iscA appeared to be required at high concentrations of oxygen (37).…”

Fe-S Cluster Assembly Pathways in Bacteria

“…The inability of C99S and C101S IscA mutants to bind iron showed that a cysteine pocket may form the binding site for a monoiron center (see below) (28). Recent studies also indicated that L-cysteine could mobilize the iron center in IscA for Fe-S cluster assembly on IscU, whereas biologically related thiols such as N-acetyl-L-cysteine or reduced glutathione failed to mobilize the iron center (25). Based on these experiments, IscA was proposed to act as an iron chaperone that binds free iron for donation to IscU (132).…”